Engineering Research Center for Aquatic Products Processing of Fujian Province

Xiamen, China

Engineering Research Center for Aquatic Products Processing of Fujian Province

Xiamen, China
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Li M.-S.,Jimei University | Li M.-S.,Engineering Research Center for Aquatic Products Processing of Fujian Province | Li T.,Jimei University | Li T.,Engineering Research Center for Aquatic Products Processing of Fujian Province | And 12 more authors.
Biochemical Engineering Journal | Year: 2017

The myofibril-bound serine proteinase (MBSP) from the skeletal muscle of fish belongs to a class of trypsin-like serine proteinases with higher thermal stability than trypsin. To investigate the mechanism responsible for the thermal stability of MBSP from Crucian carp, mutagenesis of key residues were identified by amino acid sequence alignments and Protein Mutant Stability Prediction online prediction software. Meanwhile, five mutants (MutP95T, MutR125N, MutA127S, MutI130D, and MutA127S/I130D) were obtained using site-directed mutagenesis. The mutants and wild-type enzymes were expressed in Pichia pastoris (SMD1168), the secondary structure of the mutants of MBSP was approximately similar to that of wild-type MBSP by circular dichroism spectroscopy analysis. The thermal Inactivation parameters showed that the half-life for thermal inactivation (t1/2) of MutP95T was reduced by approximately 10 min, whereas MutA127S, MutI130D and MutA127S/I130D were more stable than the wild-type and increased by approximately 18 min, 15 min and 24 min, respectively. The unfolding free energy difference (ΔΔG) between the variants and wild-type of MutA127S, MutI130D and MutA127S/I130D was enhanced when compared with the wild-type. As a result, the mutants were efficiently produced with altered thermal stability, and MBSP could potentially be used as novel tool enzyme for mass spectrometric identification. © 2017 Elsevier B.V.


Weng L.,Jimei University | Weng L.,Engineering Research Center for Aquatic Products Processing of Fujian Province | Lin Y.,Jimei University | Zhang L.,Jimei University | And 6 more authors.
Journal of Chinese Institute of Food Science and Technology | Year: 2015

Prolyl endopeptidase (PEP) is a kind of endopeptidase which specifically cleaves low molecular weight peptides on the carboxyl side of proline residues, plays an important role in further degradation of collagens. In the present study, a prolyl endopeptidase was purified to homogeneity from grass carp muscle by ammonium sulfate fractionation, column chromatographies including DEAE-Sephacel, Phenyl-Sepharose and Hydroxyapatite and the biochemical characteristics were investigated. SDS-PAGE showed that the molecular weight of PEP was 75 ku. Optimal pH of the purified enzyme was 6.0, and it was stable at pH between 5.5 to 7.5. The optimal temperature of the enzyme was 30℃ while with poor thermal stability. The enzyme activity was strongly inhibited by SUAM, a competitive and specific inhibitor of prolyl endopeptidases. PEP showed it specifically cleaved fluorogenic substrates having proline residue on the carboxyl sides, strongly suggesting that enzyme is a kind of prolyl endopeptidase. The Km and kcat of the enzyme was 19.23 μmol/L and 2.5 s-1. MALDI-TOF-MS analysis revealed that the sequence of grass carp PEP shared 98.3%, 96.1% and 94.4% identities to PEPs from Oryzias latipes, Oreochromis niloticus and Danio rerio, respectively. ©, 2015, Chinese Institute of Food Science and Technology. All right reserved.

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